Converted methylated dna

Positive Control. Each run must contain two positive controls that consist of methylated DNA. The CpGenome Universal Methylated DNA (Millipore) has to be diluted to 10 ng/uL for optimal results and bisulfite converted. The Converted Methylated DNA (Promega) needs to be diluted to 3 ng/μL.

Negative Control: Each run must contain one negative control that consists of unmethylated DNA. The CpGenome Universal Unmethylated DNA (Millipore) has to be diluted to 10 ng/μLfor optimal results and bisulfite converted.

No Template Control (NTC). Each run must contain a no template control (NTC) that consists of all reagents with the exception of any DNA sample.

In 5 clean labeled 0.2 mL PCR tubes, make 5 serial dilutions 1:5 of the converted CpGenome Universal Methylated DNA as shown in Table 2.

CpGenome Universal Methylated DNA was converted in the previous step.

In two clean labeled 1.5 mL microcentrifuge tube prepare the MGMT and ACTB master mixes as shown in Table 3.

Add 16 mcl of the MGMT Master Mix to the appropriate wells of a MicroAmp® Fast Optical 96-well Reaction Plate.

Add 16 mcl of the ACTB Master Mix to the appropriate wells of a MicroAmp^® Fast Optical 96-well Reaction Plate.

Add 4 mcl of standard control to the appropriate wells of the 96-well plate.

Mix the volume by pipetting up and down.

Add 4 mcl of methylated control, unmethylated control, converted control and samples to the appropriate wells of the 96-well plate.

Seal the plate with a MicroAmp^® Optical Adhesive Film.

Briefly centrifuge the 96-well plate to ensure that all volume is collected in the bottom of the wells and that no bubbles are present.